Abstract
Voltage-gated Ca2+ channels (VGCCs) mediate the influx of Ca2+ that regulates many cellular events, and mutations in VGCC genes cause serious hereditary diseases in mammals. The yeast Saccharomyces cerevisiae has only one gene encoding the putative pore-forming alpha1 subunit of VGCC, CCH1. Here, we identify a cch1 allele producing a completely nonfunctional Cch1 protein with a Gly1265 to Glu substitution present in the domain III S2-S3 cytoplasmic linker. Comparison of amino acid sequences of this linker among 58 VGCC alpha1 subunits from 17 species reveals that a Gly residue whose position corresponds to that of the Cch1 Gly1265 is completely conserved from yeasts to humans. Systematic amino acid substitution analysis using 10 amino acids with different chemical and structural properties indicates that the Gly1265 is essential for Cch1 function because of the smallest residue volume. Replacement of the Gly959 residue of a rat brain Cav1.2 alpha1 subunit (rbCII), positionally corresponding to the yeast Cch1 Gly1265, with Glu, Ser, Lys, or Ala results in the loss of Ba2+ currents, as revealed by the patch clamp method. These results suggest that the Gly residue in the domain III S2-S3 linker is functionally indispensable from yeasts to mammals. Because the Gly residue has never been studied in any VGCC, these findings provide new insights into the structure-function relationships of VGCCs.
Highlights
Voltage-gated Ca2ϩ channels (VGCCs)5 in the plasma membrane mediate the influx of Ca2ϩ that serves as the second messenger of electrical signals to initiate many cellular events, including muscle contraction, neurotransmitter release, and gene expression [1]
Replacement of the Gly959 residue of a rat brain Cav1.2 ␣1 subunit, positionally corresponding to the yeast Cch1 Gly1265, with Glu, Ser, Lys, or Ala results in the loss of Ba2؉ currents, as revealed by the patch clamp method. These results suggest that the Gly residue in the domain III S2–S3 linker is functionally indispensable from yeasts to mammals
We have shown the importance of the Gly residue present in the domain III S2–S3 linker of the yeast putative VGCC ␣1 subunit Cch1 and of the rat VGCC ␣1 subunit rbCII, a member of Cav1.2
Summary
Yeast Strains and Media—The parental strain H207 (MATa his3-⌬1 leu112 trp289 ura sst1–2) and its derivative mutant strains H3031 (MATa cch (formerly mid3-1) his3-⌬1 leu112 trp289 ura sst1–2) and H314 (MAT␣ cch1⌬::TRP1 his3-⌬1 leu112 trp289 ura sst1–2) were described previously [13, 17]. To construct pCCH1D, a pUC18-derived plasmid carrying PCCH1 and TCCH1, the PCR-amplified promoter and terminator fragments of the CCH1 gene of H207 were cut with appropriate restriction enzymes and inserted into the multicloning site of pUC18, an adaptor, which contains a stop codon and an XhoI site, was inserted between PCCH1 and TCCH1. To construct pBCT111, PCCH1 and TCCH1 of pBCS111 were replaced with the PTDH3-containing 0.7-kb EcoRI-BamHI fragment of pUGPD [29] and the TADH1-containing 0.3-kb PstI-SphI fragment of pGBKT7 (Clontech, Palo Alto, CA), respectively. The plasmids, pBCT-CCH1H-EGFP or pBCT-CCH1Hm1-EGFP, used to express EGFP-tagged Cch proteins were constructed by inserting the 0.6-kb NcoI (blunted)-NotI fragment of pEGFP (Clontech) between the SalI (blunted) and NotI sites of pBCTCCH1H or pBCT-CCH1Hm1, respectively.
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